WO2022131667A1 - Pharmaceutical composition, for enhancing melanoma treating effect, comprising oligodendrocyte transcription factor 2 inhibitor as active ingredient - Google Patents
Pharmaceutical composition, for enhancing melanoma treating effect, comprising oligodendrocyte transcription factor 2 inhibitor as active ingredient Download PDFInfo
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- WO2022131667A1 WO2022131667A1 PCT/KR2021/018584 KR2021018584W WO2022131667A1 WO 2022131667 A1 WO2022131667 A1 WO 2022131667A1 KR 2021018584 W KR2021018584 W KR 2021018584W WO 2022131667 A1 WO2022131667 A1 WO 2022131667A1
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- transcription factor
- olig2
- inhibitor
- pharmaceutical composition
- melanoma
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Definitions
- the present invention relates to a pharmaceutical composition for enhancing the therapeutic effect of melanoma, and more particularly, a composition for enhancing the melanoma therapeutic effect of dabrafenib comprising an inhibitor of expression or activity of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) is about
- Skin cancer is a malignant tumor of the skin, and representative types include basal cell carcinoma, squamous cell carcinoma, and melanoma. Among them, melanoma can occur in any area where melanocytes exist, such as the skin, eyeballs, and cerebrospinal fluid membranes. known as a tumor.
- melanoma The main site of metastasis of melanoma is the skin other than the primary lesion, but any other organs such as lymph nodes, bones, lungs, liver, and spleen can also be involved.
- melanoma has high resistance to conventional chemotherapeutic agents and radiation, and therefore is an intractable disease for which there is no special treatment method to date when abnormally advanced or recurrent.
- Transcription factor Olig2 is a transcription factor expressed in the central nervous system during embryonic development and is known to play a role in regulating the differentiation and cell cycle progression of oligodendrocytes (oligodendrocytes) and promoting the growth of brain tumors. Transcription factor Olig2 was confirmed to be overexpressed in cells other than oligodendroglioma (oligodendroglioma), such as lung cancer, breast cancer, and melanoma (PCT application number: 10-2017-7027034). The effect on the efficacy of therapeutic agents has not yet been specifically studied.
- the present inventors have made research efforts to discover substances for treating melanoma.
- dabrafenib When treated in combination with dabrafenib, it was confirmed that the growth of melanoma cells was more effectively inhibited than when dabrafenib alone was treated.
- the present invention was completed by confirming the fact that the recurrence of melanoma patients administered with brafenib can also be suppressed.
- an object of the present invention is to provide a pharmaceutical composition for preventing or treating melanoma, comprising an inhibitor of BRAF inhibitor and Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression or activity.
- Olig2 Oledendrocyte transcription factor 2
- the present invention provides a pharmaceutical composition for enhancing the effect of preventing or treating melanoma used in combination with a BRAF inhibitor, wherein the composition comprises an inhibitor of the expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient.
- Olig2 Oligendrocyte transcription factor 2
- the present invention includes a BRAF inhibitor and an Olig2 (Oligodendrocyte transcription factor 2) gene or inhibitor of expression or activity of a gene or protein, preventing or treating melanoma; Or it provides a pharmaceutical composition for preventing or treating metastasis of melanoma.
- Olig2 Oligodethelial growth factor 2
- the BRAF inhibitor may be dabrafenib.
- the present invention provides a method for preventing or treating melanoma in combination with a BRAF inhibitor; Or as a pharmaceutical composition for preventing or enhancing the effect of metastasis of melanoma, the composition provides a pharmaceutical composition comprising an inhibitor of expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient .
- Olig2 Oligendrocyte transcription factor 2
- the BRAF inhibitor may be dabrafenib.
- the transcription factor Olig2 (Oligodendrocyte transcription factor 2) inhibitor is an antisense oligonucleotide specific for the transcription factor Olig2 (Oligodendrocyte transcription factor 2), small interfering RNA (siRNA), short hairpin It may be any one selected from the group consisting of RNA (short hairpin RNA; shRNA) and ribozyme.
- the transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein activity inhibitor is a transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein that specifically binds to a protein, peptide, peptide mimetics, It may be any one selected from the group consisting of aptamers, antibodies, and natural products.
- the combination may be administered simultaneously (simultaneous), separately (separate) or sequentially (sequential).
- the Olig2 expression inhibitor when administered in combination with dabrafenib, it is confirmed that the melanoma treatment effect of dabrafenib is enhanced. is expected to be useful.
- Figure 2a is the result of confirming the cell proliferation rate in A375 cells in which the expression of Olig2 is suppressed.
- Figure 2b is the result of confirming the cell proliferation rate in 501mel cells in which the expression of Olig2 is suppressed.
- Figure 2c is a result of confirming the number of cells through cell count analysis in A375 cells in which Olig2 expression is suppressed.
- Figure 2d is a result of confirming the number of cells through cell count analysis in 501mel cells in which the expression of Olig2 is suppressed.
- 3a is a result of microscopic observation of cell migration over 24 hours and 48 hours in A375 cells in which Olig2 expression is suppressed.
- Figure 3b is the result of quantification of the difference in the area of the wound gap according to the lapse of 24 hours and 48 hours in A375 cells in which the expression of Olig2 is suppressed compared to the control group.
- Figure 4a is the result of observing the cell migration according to the lapse of 24 hours and 48 hours in 501mel cells in which the expression of Olig2 is suppressed under a microscope.
- Figure 4b is the result of quantification of the difference in the area of the wound gap according to the lapse of 24 hours and 48 hours in 501mel cells in which Olig2 expression is suppressed compared to the control group.
- 5a is a result of confirming cell mobility using a transwell, and is a result of staining A375 cells that migrated through the pores of the chamber with DAPI and observing it under a microscope.
- Figure 5b is the result of counting and quantifying the nuclei stained by using the Image J program of the migrated A375 cells.
- 6a is a result of confirming cell invasiveness using a transwell, and is a result of staining A375 cells infiltrating through Matrigel instead of an extracellular matrix with DAPI and observing it under a microscope.
- Figure 6b is the result of counting and quantifying the stained nucleus of the infiltrating A375 cells using the Image J program.
- the present inventors used an inhibitor of expression or activity of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) and a targeted therapeutic agent, dabrafenib.
- Olig2 the transcription factor 2
- dabrafenib a targeted therapeutic agent
- the present invention provides a pharmaceutical composition for preventing or treating melanoma, comprising an inhibitor of BRAF and Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression or activity.
- the term "BRAF inhibitor” may be dabrafenib, a targeted therapeutic agent used for the treatment of late-stage melanoma, and in the present invention, dabrafenib is known to regulate cell growth.
- dabrafenib By suppressing the expression of BRAF, a gene encoding the B-Raf protein, it is being used to treat melanoma and metastatic non-small cell lung cancer.
- the present invention provides a pharmaceutical composition for enhancing the effect of preventing or treating melanoma used in combination with a BRAF inhibitor, the composition comprising an inhibitor of expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient It provides a pharmaceutical composition, characterized in that.
- Olig2 Oligendrocyte transcription factor 2
- protein may be used synonymously with “polypeptide” or “polypeptide”, and refers to a polymer of amino acid residues. Proteins apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.
- the transcription factor Olig2 (Oligodendrocyte transcription factor 2, hereinafter Olig2) gene or protein expression or activity inhibitor refers to a substance that inhibits or interferes with the function of Olig2. Such inhibition or interference may be achieved by interfering with the transcription of the Olig2 gene or by inhibiting the translation of the mRNA of the Olig2 gene. Alternatively, the activity of the Olig2 protein may be reduced or inactivated by specifically binding to the active site of the Olig2 protein or by causing a protein structural modification.
- transcription factor Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression inhibitor) is not limited thereto, but transcription factor Olig2 (Oligendrocyte transcription factor 2 mRNA specific antisense oligonucleotide, small interfering RNA) (small interfering RNA; siRNA), short hairpin RNA (shRNA), and ribozyme may be any one selected from the group consisting of.
- transcription factor Olig2 Oligendrocyte transcription factor 2 mRNA specific antisense oligonucleotide, small interfering RNA) (small interfering RNA; siRNA), short hairpin RNA (shRNA), and ribozyme may be any one selected from the group consisting of.
- the inhibitor of the activity may refer to a substance that makes the same type of activity lower than the intrinsic activity that the non-genetically engineered parent cell (eg, wild-type) does not have or has.
- the term "inhibitor of transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein activity" of the present invention is not limited thereto, a compound, peptide, or peptide mimetic that specifically binds to transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein It may be any one selected from the group consisting of s, aptamers, antibodies, and natural products.
- nucleotides are the building blocks of oligonucleotides and polynucleotides, and for purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides.
- nucleotides such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (absent in nucleosides). Nucleosides and nucleotides may also be referred to interchangeably as “unit” or “monomer.”
- Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U).
- Oligonucleotides may include nucleosides with modified nucleobases. For example, 5-methyl cytosine is often used instead of cytosine.
- complementarily binding may include Watson-Crick base-binding between unmodified nucleobases and modified nucleobases (see, e.g., Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).
- the antisense nucleotide is defined as an oligonucleotide capable of regulating the expression of a target gene by hybridizing to a target nucleic acid, in particular, to an adjacent sequence on the target nucleic acid.
- Antisense oligonucleotides are not inherently double-stranded and therefore not siRNA or shRNA.
- the small interfering RNA refers to RNA capable of inducing RNAi (RNA interference) that inhibits gene activity.
- the small interfering RNA may be a miRNA or siRNA capable of inhibiting the expression of the Olig2 gene, and the small interfering RNA may have any form as long as it inhibits the expression or activity of the Olig2 gene.
- siRNA obtained by chemical synthesis, biochemical synthesis, or in vivo synthesis, or double-stranded RNA of 10 bases or more in which double-stranded RNA of about 40 bases or more is degraded in the body, etc. can be used.
- the "aptamer” refers to a nucleic acid molecule having binding activity to a predetermined target molecule.
- the aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be in a linear or cyclic form.
- the shorter the nucleotide sequence constituting the aptamer the shorter the chemical synthesis and mass production. It is known that it is easier, has excellent cost advantages, is easy to chemically modify, has excellent in vivo stability, and has low toxicity.
- the “antibody” refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody is a marker gene by cloning each gene into an expression vector according to a conventional method.
- the protein encoded by The form of the antibody is not particularly limited, and any part thereof is included in the antibody of the present invention as long as it has polyclonal antibody, monoclonal antibody or antigen-binding property, and all immunoglobulin antibodies may be included as well as humanized antibody, etc. It may also contain special antibodies of
- the antibody includes functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains.
- a functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2 and Fv.
- the term "combination” refers to co-administration, but is not limited thereto, but the Olig2 inhibitor of the present invention and dabrafenib are administered in combination simultaneously (simultaneous), separately (separate) or sequentially (sequential). it could be
- the present inventors confirmed that the preventive or therapeutic effect of melanoma was improved when the oilg2 gene or protein expression or activity inhibitor and dabrafenib were administered in combination through specific examples.
- melanoma growth can be inhibited by enhancing the effect of dabrafenib when treating melanoma cells in which the expression of Olig2 is suppressed, dabrafenib, a melanoma therapeutic agent. (see Example 4).
- the inventors of the present invention specifically confirmed that when an inhibitor capable of inhibiting the expression or activity of Olig2 of the present invention is treated, metastasis and growth of melanoma can be significantly inhibited.
- the inhibitor and dabrafenib were administered in combination, it was confirmed that even a small amount could enhance the melanoma inhibitory effect of dabrafenib.
- the Olig2 inhibitor can be widely used in the field of melanoma treatment.
- the pharmaceutical composition may include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.
- the pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added.
- diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
- the pharmaceutical composition may be administered in combination parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intradermally or orally.
- the drug may be administered orally or directly to a target organ, and the dosage may vary depending on the patient's age, sex, health condition and weight, type of concurrent treatment, administration time, administration method, excretion rate, disease severity, and frequency of treatment. and the nature of the desired effect.
- it may be administered systemically or locally in consideration of conditions such as the disease to be treated, the need for site-specific treatment, and the amount of the drug to be administered.
- the pharmaceutical composition may have any pharmaceutical formulation.
- a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulation, solution, or suspension in a form suitable for parenteral injection as a sterile solution, suspension or emulsion, or in a form suitable for topical administration as an ointment or cream. It may be in a suitable form or may be in a form suitable for rectal administration as a suppository.
- the composition may be a tablet, pill, injection, or a combination thereof.
- the pharmaceutical composition may be in unit dosage form suitable for single administration of precise dosages.
- the pharmaceutical composition may be used alone or in combination, or in combination with other therapeutic agents or diagnostic agents.
- the pharmaceutical composition may be administered concurrently with other agents typically prescribed for the intended disease in accordance with generally accepted medical practice.
- the pharmaceutical composition can generally be used in a mammal, for example, a human in vivo (in vivo).
- the present invention provides a method for preventing or treating melanoma, comprising administering a BRAF inhibitor and an Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression or activity inhibitor to an individual in need thereof.
- Olig2 Oledendrocyte transcription factor 2
- the term “administration” means providing a given composition of the present invention to a subject by any suitable method.
- the term "subject” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, dogs, cats, horses, and cattle. means mammals.
- Another aspect provides the use of a BRAF inhibitor and an Oligodendrocyte transcription factor 2 (Olig2) gene or inhibitor of expression or activity for the manufacture of a medicament for the prophylaxis or treatment of melanoma.
- Olig2 Oligodendrocyte transcription factor 2
- Human melanoma cells, A735, 501mel and HM3KO cells were treated with Dulbecco's modified eagle supplemented with 10% Fetal Bovine Serum (Gibco, Los Angeles, CA, USA) and 1% penicillin/streptomycin.
- Culture in ⁇ s medium (DMEM; Welgene Inc., Korea).
- MNT1 a human melanoma cell, was prepared in minimum essential media (MEM; Welgene Inc.) supplemented with 20% FBS, 1% penicillin/streptomycin and 20 mM hydroxyethyl piperazineethanesulfonic acid (HEPES). Korea).
- NHEMs Normal human melanocytes, NHEMs, were prepared from Human Melanocyte Growth Supplement (HMGS; Invitrogen Life Technologies, Carlsbad, CA, USA) and medium 254 (Medium 254, M-254) containing 1% penicillin/streptomycin. -500; Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified atmosphere of 37° C. and 5% CO 2 .
- Ez-Cytox assay kit (Daeil Lab Service Co Ltd, Seoul, Korea) was used. Specifically, 10 ⁇ l of Ez-Cytox solution was added to each well-plate, and then the well-plate was added to 5% Incubated for an additional 1 hour under CO 2 conditions and in an incubator at 37°C. Cell viability was confirmed at 490 nm wavelength by an ELISA reader (Tecan, Mannedorf, Switzerland), and relative cytotoxicity was measured as a percentage of cell viability.
- Cells were seeded in a 96-well plate at 1 ⁇ 10 4 cells/well, and then transfected using CRISPR/Cas9 plasmid. After 72 hours, the cells were trypsin-treated. 10 ul of trypan blue dye (Sigma Aldrich, St. Louis, MO, USA) was mixed in the same manner as the cell suspension. After staining the cells with trypan blue dye, each sample was repeated three times and the number of cells was counted using a hemocytometer.
- trypan blue dye Sigma Aldrich, St. Louis, MO, USA
- RNA total RNA
- melanoma cells prepared in Example 1-1 were treated with TRIZOL reagent (Takara Bio, Inc., Tokyo, Japan), and then 1/5 of TRIZOL in chloroform (Chloroform, CHCl 3 , 99%, Junsei Chemical, Japan) was treated and mixed.
- TRIZOL reagent Tekara Bio, Inc., Tokyo, Japan
- chloroform Chloroform, CHCl 3 , 99%, Junsei Chemical, Japan
- After mixing the Vortex mixer centrifuged for 15 minutes at 13,000 rpm at a temperature of 4 °C. A certain amount of the transparent supernatant was taken and mixed with the same amount of isopropanol. After inverting the tube centrifuge at 4° C., 13,000 rpm, 20 min. The supernatant was removed and the remaining pellet was diluted with diethyl pyrocarbonate treated water.
- cDNA complementary DNA
- RT-PCR reverse transcription polymerase chain reaction
- melanoma cells were seeded in 12 well plates and then cells were trypsinized 24 h after transfection with CRISPR/Cas9 plasmids.
- Cells were re-seeded with polycarbonate membranes (Corning Incorporated, Corning, NY, USA) with 8.0 ⁇ m pore size in DMEM medium containing 1% FBS at 5 x 10 4 cells per chamber, followed by 18 h. incubated for a while.
- the upper chamber was coated with Matrigel® (Corning, Kaiserslautern, Germany) diluted with serum-free DMEM medium, and the bottom of the upper chamber was coated with 1% gelatin.
- the bottom chamber was filled with DMEM medium containing 10% FBS.
- DMEM medium containing 10% FBS.
- Cells that invaded through the Matrigel-coated membrane were fixed in 10% formalin and washed twice with PBS.
- DAPI 4',6-diamidino-2-phenylindole
- Invasive cells were detected by fluorescence microscopy at x100 magnification. Stained nuclei were counted and quantified using ImageJ software 1.45s (US National Institutes of Health, Bethesda, MD, USA).
- CRISPR-Cas9 technique was used in 501mel cells, a malignant melanoma cell, to prepare cells in which the expression of the transfer factor Olig 2 (Oligodendrocyte transcription factor 2) was suppressed.
- UltraCruz transfection reagent UltraCruz Transfection Reagent (Santacruz Biotechnology, Dallas, TX, USA)
- 20nt guide sgRNA and Cas9 nuclease encoding three The sc-400670-KO-2 consisting of different gRNA plasmids: Olig2 CRISPR/Cas9 KO plasmids (h2) (Santacruz Biotechnology, Dallas, TX, USA) were transfected.
- the three gRNAs of the plasmid may be gRNA1: 5'-GGCTGCGTCGTCCACCAAGA-3', gRNA2: 5'-CACCTCGTCGTCTACGTCGT-3' or gRNA3: 5'-CGCCCATAGCCGACACGTGG-3'.
- cancer cells have a characteristic of infinitely proliferating unlike normal cells
- the proliferation rate of malignant melanoma cells in which Olig2 expression is suppressed was measured. More specifically, in the malignant melanoma cells (A375 cells and 501mel cells) in which the expression of Olig2 prepared in Example 2 is suppressed, Ez-CyToX cell viability was analyzed according to the concentration and time of treatment with Olig2 gRNA.
- Metastasis one of the main characteristics of malignant melanoma, is a phenomenon in which cells are separated from the primary tumor and move around. Migrating melanoma cells circulate in blood and lymph vessels, settle in other end organs, form secondary tumors, and eventually metastasize. (NGUYEN, DX, BOS, PD and MASSAGUE, J., 2009. Metastasis: from dissemination to organ-specific colonization. Nature Reviews Cancer, 9 (4), pp. 274.)
- FIGS. 3a A375 cells
- 4a 501 mel cells
- transwell metastasis assay was performed to analyze cell mobility.
- Cells were seeded into the upper chamber and allowed to migrate through the transwell chamber for 18 hours. Thereafter, the cells that migrated through the pores of the chamber were stained with DAPI and observed under a microscope (see Fig. 5a), and the stained nuclei were counted and quantified using the Image J program.
- Fig. 5b 18 As time elapsed, it was confirmed that a 63 ⁇ 5.2% decrease in the group treated with 1 ⁇ g of gRNA for Olig2 inhibition and 58 ⁇ 5.0% decreased in the group treated with 2 ⁇ g of gRNA.
- matrigel was diluted with 0% FBS DMEM medium, and then coated on the upper chamber. was stained with and observed under a microscope (see Fig. 6a), and the stained nuclei were counted and quantified using the Image J program. It was confirmed that the decrease was 48 ⁇ 8.2% in the group treated with 1 ⁇ g, and decreased by 54 ⁇ 8.6% in the group treated with gRNA 2 ⁇ g.
- the present inventors confirmed that when Olig2 is inhibited through the above results, metastasis of melanoma can be inhibited to a significant level.
- Example 4 Confirmation of survival rate of malignant melanoma cells when oligodendrocyte transcription factor 2 expression is suppressed by Dabrafenib treatment
- Dabrafenib a melanoma treatment previously approved by the US FDA as a B-Raf inhibitor
- a B-Raf inhibitor a melanoma treatment previously approved by the US FDA as a B-Raf inhibitor
- the cell viability was checked.
- FIG. 7 and Table 3 below, when dabrafenib was administered to malignant melanoma cells in which Olig2 expression was suppressed, and when dabrafenib was treated to Olig2-expressing melanoma cells It was confirmed that the growth inhibitory effect of melanoma cells was superior.
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Abstract
The present invention relates to a pharmaceutical composition for enhancing melanoma metastasis treating effect. More particularly, it has been confirmed that the melanoma preventing or treating effect of dabrafenib was notably enhanced when the composition comprising the oligodendrocyte transcription factor 2 (OLIG2) inhibitor was concomitantly administered with dabrafenib. The pharmaceutical composition of the present invention enables effective treatment of melanoma even when less dabrafenib is used than an existing dose, and thus can be free from the problem of toxicity and side effects.
Description
본 발명은 흑색종 치료 효과 증진용 약학적 조성물에 관한 것으로서, 보다 구체적으로 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 또는 활성 억제제를 포함하는 다브라페닙(dabrafenib)의 흑색종 치료 효과 증진용 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for enhancing the therapeutic effect of melanoma, and more particularly, a composition for enhancing the melanoma therapeutic effect of dabrafenib comprising an inhibitor of expression or activity of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) is about
야외활동 증가와 같은 최근 생활 패턴 및 환경오염에 따른 오존층의 파괴로 인한 자외선의 유입량 증가로 인해 각종 유해물질이나 자외선의 노출 기회가 빈번해지고, 인간의 평균수명이 늘어나면서 자외선 축적량이 많은 고령 인구수가 늘어남에 따라 피부암과 관련한 환자수가 전 세계적으로 지속해서 증가하고 있다. 피부암은 피부 악성 종양으로 대표적인 종류로는 기저세포암, 편평상피세포암, 흑색종이 있다. 그 중 흑색종은 피부, 눈알, 뇌척수 연막 등 멜라닌 세포가 존재하는 어느 부위에서나 발생할 수 있으며, 다른 피부암과 달리 성장 속도가 매우 빨라 발생 초기에 다른 장기로 전이율이 높고 재발하기 쉬워 악성도가 높은 종양으로 알려져 있다.Due to the increase in the amount of ultraviolet radiation due to the destruction of the ozone layer due to recent living patterns such as the increase in outdoor activities and environmental pollution, the exposure to various harmful substances and ultraviolet rays is becoming more frequent. As the number of skin cancer-related patients increases, the number of patients worldwide continues to increase. Skin cancer is a malignant tumor of the skin, and representative types include basal cell carcinoma, squamous cell carcinoma, and melanoma. Among them, melanoma can occur in any area where melanocytes exist, such as the skin, eyeballs, and cerebrospinal fluid membranes. known as a tumor.
흑색종의 주요 전이 부위는 원발병소 이외의 피부이지만 그 밖에도 림프절, 뼈, 폐, 간, 비장 등 어떤 기관들도 침범될 수 있다. 특히 흑색종은 종래의 화학요법제 및 방사선에의 높은 저항성을 가지기 때문에 초기 이상 진행되거나 재발한 경우에는 현재까지 특별한 치료방법이 없는 난치성 질환이다.The main site of metastasis of melanoma is the skin other than the primary lesion, but any other organs such as lymph nodes, bones, lungs, liver, and spleen can also be involved. In particular, melanoma has high resistance to conventional chemotherapeutic agents and radiation, and therefore is an intractable disease for which there is no special treatment method to date when abnormally advanced or recurrent.
전사인자 Olig2는 배아 발달기 동안 중추신경계통에서 발현되는 전사 인자로 oligodendrocyte(희소돌기신경교)의 분화와 세포 주기 진행을 조절하며 뇌종양의 성장을 촉진하는 역할을 하는 것으로 알려져 있다. 전사인자 Olig2는 oligodendroglioma(희소돌기아교세포종) 이외에 폐암, 유방암, 흑색종 등의 세포에서 과발현하는 것으로 확인되었으나(PCT 출원번호: 10-2017-7027034), 전사인자 Olig2를 기존 치료제와 병용하는 경우 기존 치료제의 효능에 미치는 영향에 대해서는 아직 구체적으로 연구된 바가 없다. Transcription factor Olig2 is a transcription factor expressed in the central nervous system during embryonic development and is known to play a role in regulating the differentiation and cell cycle progression of oligodendrocytes (oligodendrocytes) and promoting the growth of brain tumors. Transcription factor Olig2 was confirmed to be overexpressed in cells other than oligodendroglioma (oligodendroglioma), such as lung cancer, breast cancer, and melanoma (PCT application number: 10-2017-7027034). The effect on the efficacy of therapeutic agents has not yet been specifically studied.
상기와 같은 배경하에, 본 발명자들은 흑색종을 치료하기 위한 물질을 발굴하기 위해 연구 노력한 결과, 전사인자 Olig2(Oligodendrocyte transcription factor 2, 이하 Olig2)의 억제제와 흑색종의 표적치료제로 알려진 다브라페닙(dabrafenib)을 병용하여 처리할 경우 다브라페닙만을 단독으로 처리하는 경우 보다 흑색종 세포의 생장을 효과적으로 억제하는 것을 확인함으로써 Olig2의 억제제가 다브라페닙에 의한 흑색종 치료효과를 증진시킬 수 있고, 다브라페닙 투여한 흑색종 환자의 재발도 억제할 수 있다는 사실을 확인함으로써 본 발명을 완성했다.Under the above background, the present inventors have made research efforts to discover substances for treating melanoma. As a result, dabrafenib ( When treated in combination with dabrafenib), it was confirmed that the growth of melanoma cells was more effectively inhibited than when dabrafenib alone was treated. The present invention was completed by confirming the fact that the recurrence of melanoma patients administered with brafenib can also be suppressed.
이에, 본 발명은 BRAF 억제제 및 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 또는 활성 억제제를 포함하는, 흑색종의 예방 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating melanoma, comprising an inhibitor of BRAF inhibitor and Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression or activity.
또한, 본 발명은 BRAF 억제제와 병용되는 흑색종 예방 또는 치료 효과 증진용 약학적 조성물로서, 상기 조성물은 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 또는 활성 억제제를 유효성분으로 포함하는 것을 특징으로 하는, 약학적 조성물을 제공하는 것을 다른 목적으로 한다.In addition, the present invention provides a pharmaceutical composition for enhancing the effect of preventing or treating melanoma used in combination with a BRAF inhibitor, wherein the composition comprises an inhibitor of the expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient. Another object is to provide a pharmaceutical composition.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
상기 목적을 달성하기 위해, 본 발명은 BRAF 억제제 및 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 또는 활성 억제제를 포함하는, 흑색종의 예방 또는 치료; 또는 흑색종의 전이 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention includes a BRAF inhibitor and an Olig2 (Oligodendrocyte transcription factor 2) gene or inhibitor of expression or activity of a gene or protein, preventing or treating melanoma; Or it provides a pharmaceutical composition for preventing or treating metastasis of melanoma.
본 발명의 일 구현예에 있어서, 상기 BRAF 억제제는 다브라페닙(dabrafenib)일 수 있다.In one embodiment of the present invention, the BRAF inhibitor may be dabrafenib.
또한, 본 발명은 BRAF 억제제와 병용되는 흑색종 예방 또는 치료; 또는 흑색종의 전이 예방 또는 치료 효과 증진용 약학적 조성물로서, 상기 조성물은 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 또는 활성 억제제를 유효성분으로 포함하는 것을 특징으로 하는, 약학적 조성물을 제공한다.In addition, the present invention provides a method for preventing or treating melanoma in combination with a BRAF inhibitor; Or as a pharmaceutical composition for preventing or enhancing the effect of metastasis of melanoma, the composition provides a pharmaceutical composition comprising an inhibitor of expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient .
본 발명의 일 구현예에 있어서, 상기 BRAF 억제제는 다브라페닙(dabrafenib)일 수 있다.In one embodiment of the present invention, the BRAF inhibitor may be dabrafenib.
본 발명의 다른 구현예에 있어서, 상기 전사인자 Olig2(Oligodendrocyte transcription factor 2) 억제제는 전사인자 Olig2(Oligodendrocyte transcription factor 2)에 특이적인 안티센스 올리고뉴클레오티드, 작은 간섭 RNA(small interfering RNA; siRNA), 짧은 헤어핀 RNA(short hairpin RNA; shRNA) 및 리보자임(ribozyme)으로 구성된 군에서 선택되는 어느 하나일 수 있다.In another embodiment of the present invention, the transcription factor Olig2 (Oligodendrocyte transcription factor 2) inhibitor is an antisense oligonucleotide specific for the transcription factor Olig2 (Oligodendrocyte transcription factor 2), small interfering RNA (siRNA), short hairpin It may be any one selected from the group consisting of RNA (short hairpin RNA; shRNA) and ribozyme.
본 발명의 또 다른 구현예에 있어서, 상기 전사인자 Olig2(Oligodendrocyte transcription factor 2) 단백질의 활성 억제제는 전사인자 Olig2(Oligodendrocyte transcription factor 2) 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 미메틱스, 앱타머, 항체 및 천연물로 구성된 군으로부터 선택되는 어느 하나인 것일 수 있다.In another embodiment of the present invention, the transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein activity inhibitor is a transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein that specifically binds to a protein, peptide, peptide mimetics, It may be any one selected from the group consisting of aptamers, antibodies, and natural products.
본 발명의 또 다른 구현예에 있어서, 상기 병용은 동시에(simultaneous), 별도로(separate) 또는 순차적(sequential) 투여되는 것일 수 있다.In another embodiment of the present invention, the combination may be administered simultaneously (simultaneous), separately (separate) or sequentially (sequential).
본 발명에 따라 Olig2의 발현 억제제를 다브라페닙과 병용하여 투여하는 경우 다브라페닙의 흑색종 치료 효과가 증진됨을 확인함으로써 향후 악성 흑색종의 예방 및 치료 효과 증진용으로 본 발명의 Olig2의 발현 억제제가 유용하게 활용될 수 있을 것으로 기대된다.According to the present invention, when the Olig2 expression inhibitor is administered in combination with dabrafenib, it is confirmed that the melanoma treatment effect of dabrafenib is enhanced. is expected to be useful.
단, 본 발명의 효과는 상기 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.However, the effect of the present invention is not limited to the above effect, and it should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 악성 흑색종 세포인 501mel 세포의 Olig2 발현을 억제하기 위해 활용된 CRISPR-Cas9 기법의 Olig2 gRNA의 농도에 비례한 Olig2 단백질의 발현 수준을 확인한 결과이다.1 is a result of confirming the expression level of Olig2 protein in proportion to the concentration of Olig2 gRNA of the CRISPR-Cas9 technique used to suppress Olig2 expression in 501mel cells, which are malignant melanoma cells.
도 2a는 Olig2의 발현이 억제된 A375 세포에서 세포 증식률을 확인한 결과이다.Figure 2a is the result of confirming the cell proliferation rate in A375 cells in which the expression of Olig2 is suppressed.
도 2b는 Olig2의 발현이 억제된 501mel 세포에서 세포 증식률을 확인한 결과이다.Figure 2b is the result of confirming the cell proliferation rate in 501mel cells in which the expression of Olig2 is suppressed.
도 2c는 Olig2의 발현이 억제된 A375 세포에서 세포계수 분석을 통해 세포수를 확인한 결과이다.Figure 2c is a result of confirming the number of cells through cell count analysis in A375 cells in which Olig2 expression is suppressed.
도 2d는 Olig2의 발현이 억제된 501mel 세포에서 세포계수 분석을 통해 세포수를 확인한 결과이다.Figure 2d is a result of confirming the number of cells through cell count analysis in 501mel cells in which the expression of Olig2 is suppressed.
도 3a는 Olig2의 발현이 억제된 A375 세포에서 24시간 및 48시간 경과에 따른 세포 이동을 현미경으로 관찰한 결과이다.3a is a result of microscopic observation of cell migration over 24 hours and 48 hours in A375 cells in which Olig2 expression is suppressed.
도 3b는 Olig2의 발현이 억제된 A375 세포에서 24시간 및 48시간 경과에 따른 상처 간극의 면적 차이를 대조군과 비교하여 정량화한 결과이다.Figure 3b is the result of quantification of the difference in the area of the wound gap according to the lapse of 24 hours and 48 hours in A375 cells in which the expression of Olig2 is suppressed compared to the control group.
도 4a는 Olig2의 발현이 억제된 501mel 세포에서 24시간 및 48시간 경과에 따른 세포 이동을 현미경으로 관찰한 결과이다.Figure 4a is the result of observing the cell migration according to the lapse of 24 hours and 48 hours in 501mel cells in which the expression of Olig2 is suppressed under a microscope.
도 4b는 Olig2의 발현이 억제된 501mel 세포에서 24시간 및 48시간 경과에 따른 상처 간극의 면적 차이를 대조군과 비교하여 정량화한 결과이다.Figure 4b is the result of quantification of the difference in the area of the wound gap according to the lapse of 24 hours and 48 hours in 501mel cells in which Olig2 expression is suppressed compared to the control group.
도 5a는 트랜스웰(transwell)을 이용하여 세포 이동성을 확인한 결과로, 챔버의 기공을 통해 이동한 A375 세포를 DAPI로 염색하고 현미경으로 관찰한 결과이다.5a is a result of confirming cell mobility using a transwell, and is a result of staining A375 cells that migrated through the pores of the chamber with DAPI and observing it under a microscope.
도 5b는 이동한 A375 세포를 Image J 프로그램을 사용하여 염색된 핵을 계수하고 정량화한 결과이다.Figure 5b is the result of counting and quantifying the nuclei stained by using the Image J program of the migrated A375 cells.
도 6a는 트랜스웰(transwell)을 이용하여 세포 침습성을 확인한 결과로, 세포외 기질 대신 매트리젤(Matrigel)을 통해 침투된 A375 세포를 DAPI로 염색하고 현미경으로 관찰한 결과이다.6a is a result of confirming cell invasiveness using a transwell, and is a result of staining A375 cells infiltrating through Matrigel instead of an extracellular matrix with DAPI and observing it under a microscope.
도 6b는 침투한 A375 세포를 Image J 프로그램을 사용하여 염색된 핵을 계수하고 정량화한 결과이다.Figure 6b is the result of counting and quantifying the stained nucleus of the infiltrating A375 cells using the Image J program.
도 7은 501mel 세포에 Olig2 발현 억제를 위한 CRISPR-Cas9 gRNA와 다브라페닙을 각각 또는 병용하여 처리하면서 세포 생존율을 확인한 결과이다.7 is a result of confirming cell viability while treating 501mel cells with CRISPR-Cas9 gRNA and dabrafenib for suppression of Olig2 expression, respectively or in combination.
본 발명자들은 본 발명자들은 흑색종을 치료하기 위한 물질을 발굴하기 위해 연구 노력한 결과, 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 또는 활성 억제제와 표적치료제인 다브라페닙(dabrafenib)을 병용하여 처리하는 경우, 다브라페닙만을 단독으로 처리하는 경우보다 악성 흑색종 세포의 생존율을 유의하게 억제하여 다브라페닙의 흑색종 치료 효율을 증진시킨다는 사실을 확인하였는바, 이로써 본 발명을 완성하게 되었다.As a result of the present inventors' research efforts to discover a substance for treating melanoma, the present inventors used an inhibitor of expression or activity of the transcription factor Olig2 (Oligodendrocyte transcription factor 2) and a targeted therapeutic agent, dabrafenib. In this case, it was confirmed that the survival rate of malignant melanoma cells was significantly inhibited compared to the case of treatment with dabrafenib alone, thereby enhancing the melanoma treatment efficiency of dabrafenib, thereby completing the present invention.
이에, 본 발명은 BRAF 억제제 및 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 또는 활성 억제제를 포함하는, 흑색종의 예방 또는 치료용 약학적 조성물을 제공한다.Accordingly, the present invention provides a pharmaceutical composition for preventing or treating melanoma, comprising an inhibitor of BRAF and Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression or activity.
본 발명에 있어서, 용어 "BRAF 억제제"는 말기 흑색종의 치료를 위해 사용되는 표적 치료제인 다브라페닙(dabrafenib)일 수 있으며, 본 발명에 있어서 상기 다브라페닙은 세포의 성장을 조절하는 것으로 알려진 B-Raf 단백질을 암호화하는 유전자인 BRAF의 발현을 억제함으로써 흑색종 및 전이성 비소 세포성 폐암 치료에 활용되고 있다.In the present invention, the term "BRAF inhibitor" may be dabrafenib, a targeted therapeutic agent used for the treatment of late-stage melanoma, and in the present invention, dabrafenib is known to regulate cell growth. By suppressing the expression of BRAF, a gene encoding the B-Raf protein, it is being used to treat melanoma and metastatic non-small cell lung cancer.
본 발명의 다른 양태로서, 본 발명은 BRAF 억제제와 병용되는 흑색종 예방 또는 치료 효과 증진용 약학적 조성물로서, 상기 조성물은 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 또는 활성 억제제를 유효성분으로 포함하는 것을 특징으로 하는, 약학적 조성물을 제공한다.In another aspect of the present invention, the present invention provides a pharmaceutical composition for enhancing the effect of preventing or treating melanoma used in combination with a BRAF inhibitor, the composition comprising an inhibitor of expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient It provides a pharmaceutical composition, characterized in that.
본 발명에 있어서, 용어 "단백질"은 "폴리펩타이드" 또는 "폴리펩티드"와 동일한 의미로 사용될 수 있으며, 아미노산 잔기의 폴리머를 지칭한다. 단백질은 하나 이상의 아미노산 잔기가 상응하는 자연 발생한 아미노산의 인공적인 화학적 모방체인 아미노산 폴리머, 뿐만 아니라 자연 발생한 아미노산 폴리머, 변형된 잔기를 함유하는 것들, 및 비-자연 발생한 아미노산 폴리머에 적용된다.In the present invention, the term "protein" may be used synonymously with "polypeptide" or "polypeptide", and refers to a polymer of amino acid residues. Proteins apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.
상기 전사인자 Olig2(Oligodendrocyte transcription factor 2, 이하 Olig2) 유전자 또는 단백질의 발현 또는 활성 억제제는 Olig2의 기능을 저해 또는 방해하는 물질을 의미한다. 이와 같은 저해 또는 방해는 Olig2 유전자의 전사를 방해하거나, 또는 Olig2 유전자의 mRNA의 번역을 저해함으로써 이루어질 수 있다. 또는 Olig2 단백질의 활성 부위에 특이적으로 결합하거나, 단백질 구조 변형을 일으킴으로써, Olig2 단백질의 활성을 감소 또는 불활성화시킬 수 있다.The transcription factor Olig2 (Oligodendrocyte transcription factor 2, hereinafter Olig2) gene or protein expression or activity inhibitor refers to a substance that inhibits or interferes with the function of Olig2. Such inhibition or interference may be achieved by interfering with the transcription of the Olig2 gene or by inhibiting the translation of the mRNA of the Olig2 gene. Alternatively, the activity of the Olig2 protein may be reduced or inactivated by specifically binding to the active site of the Olig2 protein or by causing a protein structural modification.
본 발명에 있어서, 용어 "전사인자 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 억제제"는 이에 제한되는 것은 아니지만, 전사인자 Olig2(Oligodendrocyte transcription factor 2의 mRNA에 특이적인 안티센스 올리고뉴클레오티드, 작은 간섭 RNA(small interfering RNA; siRNA), 짧은 헤어핀 RNA(short hairpin RNA; shRNA) 및 리보자임(ribozyme)으로 구성된 군으로부터 선택되는 어느 하나인 것일 수 있다.In the present invention, the term "transcription factor Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression inhibitor" is not limited thereto, but transcription factor Olig2 (Oligendrocyte transcription factor 2 mRNA specific antisense oligonucleotide, small interfering RNA) (small interfering RNA; siRNA), short hairpin RNA (shRNA), and ribozyme may be any one selected from the group consisting of.
상기 활성의 억제제는 유전적으로 조작되지 않은 모세포(예, 야생형)가 갖지 않는 또는 갖는 내재적 활성에 비해, 동일한 타입의 활성이 보다 더 낮도록 만드는 물질을 의미할 수 있다. 상기 본 발명의 용어 "전사인자 Olig2(Oligodendrocyte transcription factor 2) 단백질의 활성 억제제"는 이에 제한되는 것은 아니지만, 전사인자 Olig2(Oligodendrocyte transcription factor 2) 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 미메틱스, 앱타머, 항체 및 천연물로 구성된 군으로부터 선택되는 어느 하나인 것일 수 있다.The inhibitor of the activity may refer to a substance that makes the same type of activity lower than the intrinsic activity that the non-genetically engineered parent cell (eg, wild-type) does not have or has. Although the term "inhibitor of transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein activity" of the present invention is not limited thereto, a compound, peptide, or peptide mimetic that specifically binds to transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein It may be any one selected from the group consisting of s, aptamers, antibodies, and natural products.
상기 뉴클레오티드는 올리고뉴클레오티드 및 폴리뉴클레오티드의 빌딩 블록이고, 본 발명의 목적을 위하여 천연 발생 및 비-천연 발생 뉴클레오티드를 둘 다 포함한다. 천연적으로, 뉴클레오티드, 예컨대 DNA 및 RNA 뉴클레오티드는 리보스 당 모이어티, 핵염기 모이어티 및 하나 이상의 포스페이트 기(뉴클레오시드에는 부재함)를 포함한다. 뉴클레오시드 및 뉴클레오티드는 또한 "단위" 또는 "단량체"와 상호교환적으로 지칭될 수 있다.Such nucleotides are the building blocks of oligonucleotides and polynucleotides, and for purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (absent in nucleosides). Nucleosides and nucleotides may also be referred to interchangeably as “unit” or “monomer.”
상기 "상보적으로 결합"하는 것의 의미는 뉴클레오시드/뉴클레오티드의 왓슨-크릭(Watson-Crick)의 염기 쌍과 관련이 있다. 왓슨-크릭 염기 쌍은 구아닌(G)-시토신(C) 및 아데닌(A)-티민(T)/우라실(U)이다. 올리고뉴클레오티드는 변형된 핵염기를 갖는 뉴클레오시드를 포함할 수 있다. 예를 들어 5-메틸 시토신이 종종 시토신 대신에 사용된다. 상기 용어 "상보적으로 결합"은 비-변형된 핵염기와 변형된 핵염기 사이의 왓슨-크릭 염기-결합을 포함할 수 있다(예를 들어, 문헌[Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055] 및 문헌[Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1] 참고).The meaning of "complementarily binding" is related to Watson-Crick base pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). Oligonucleotides may include nucleosides with modified nucleobases. For example, 5-methyl cytosine is often used instead of cytosine. The term "complementarily binding" may include Watson-Crick base-binding between unmodified nucleobases and modified nucleobases (see, e.g., Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).
상기 안티센스 뉴클레오티드는 표적 핵산, 특히 표적 핵산 상의 인접 서열에 혼성화함으로써 표적 유전자의 발현을 조절할 수 있는 올리고뉴클레오티드로 정의된다. 안티센스 올리고뉴클레오티드는 본질적으로 이중가닥이 아니고, 이에 따라 siRNA 또는 shRNA가 아니다.The antisense nucleotide is defined as an oligonucleotide capable of regulating the expression of a target gene by hybridizing to a target nucleic acid, in particular, to an adjacent sequence on the target nucleic acid. Antisense oligonucleotides are not inherently double-stranded and therefore not siRNA or shRNA.
상기 작은 간섭 RNA란, 유전자의 활성을 억제하는 RNAi(RNA interference)를 유발시킬 수 있는 RNA를의미한다. 상기 작은 간섭 RNA는 Olig2 유전자의 발현을 억제할 수 있는 miRNA, siRNA 등이 될 수 있는데, 상기 작은 간섭 RNA는 Olig2 유전자의 발현 또는 활성을 억제시키기만 하면 어떠한 형태의 것도 가능하다. 예를 들면, 화학합성 또는 생화학적 합성 또는 생체내 합성에 의해 수득되는 siRNA, 혹은 약 40개 염기 이상의 이중가닥 RNA가 체내에서 분해된 10개 염기대 이상의 이중가닥 RNA 등을 사용할 수 있다.The small interfering RNA refers to RNA capable of inducing RNAi (RNA interference) that inhibits gene activity. The small interfering RNA may be a miRNA or siRNA capable of inhibiting the expression of the Olig2 gene, and the small interfering RNA may have any form as long as it inhibits the expression or activity of the Olig2 gene. For example, siRNA obtained by chemical synthesis, biochemical synthesis, or in vivo synthesis, or double-stranded RNA of 10 bases or more in which double-stranded RNA of about 40 bases or more is degraded in the body, etc. can be used.
상기 "앱타머"는 소정의 표적 분자에 대한 결합 활성을 갖는 핵산 분자를 의미한다. 상기 앱타머는 RNA, DNA, 수식(modified) 핵산 또는 이들의 혼합물일 수 있으며, 직쇄상 또는 환상의 형태일 수 있는데, 대체로, 상기 앱타머를 구성하는 뉴클레오티드의 서열이 짧을수록 화학합성 및 대량 생산이 보다 용이하고, 비용면에서의 장점이 우수하며, 화학수식이 용이하고, 생체 내 안정성이 우수하며, 독성이 낮다고 알려져 있다.The "aptamer" refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be in a linear or cyclic form. In general, the shorter the nucleotide sequence constituting the aptamer, the shorter the chemical synthesis and mass production. It is known that it is easier, has excellent cost advantages, is easy to chemically modify, has excellent in vivo stability, and has low toxicity.
상기 "항체"란, 단백질 또는 펩티드 분자의 항원성 부위에 특이적으로 결합할 수 있는 단백질성 분자를 의미하는데, 이러한 항체는, 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 마커 유전자에 의해 코딩되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 상기 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함될 수 있을 뿐만 아니라, 인간화 항체 등의 특수 항체를 포함할 수도 있다. 아울러, 상기 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 의미하며 Fab, F(ab'), F(ab')2 및 Fv 등이 될 수 있다.The "antibody" refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody is a marker gene by cloning each gene into an expression vector according to a conventional method. The protein encoded by The form of the antibody is not particularly limited, and any part thereof is included in the antibody of the present invention as long as it has polyclonal antibody, monoclonal antibody or antigen-binding property, and all immunoglobulin antibodies may be included as well as humanized antibody, etc. It may also contain special antibodies of In addition, the antibody includes functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2 and Fv.
본 발명에 있어서, 용어 "병용"은 병용투여를 의미하는 것으로 이에 제한되는 것은 아니지만, 본 발명의 Olig2 억제제와 다브라페닙을 동시에(simultaneous), 별도로(separate) 또는 순차적(sequential)으로 병용투여하는 것일 수 있다.In the present invention, the term "combination" refers to co-administration, but is not limited thereto, but the Olig2 inhibitor of the present invention and dabrafenib are administered in combination simultaneously (simultaneous), separately (separate) or sequentially (sequential). it could be
본 발명자들은 구체적인 실시예를 통해 oilg2 유전자 또는 단백질의 발현 또는 활성 억제제와 다브라페닙을 병용하여 투여하는 경우, 흑색종의 예방 또는 치료효과가 개선되는 것을 확인하였다.The present inventors confirmed that the preventive or therapeutic effect of melanoma was improved when the oilg2 gene or protein expression or activity inhibitor and dabrafenib were administered in combination through specific examples.
본 발명의 일 실시예에서는, 흑색종 세포에 Olig2의 발현을 억제한 경우, 세포의 생존율이 유의한 수준으로 저하됨을 확인하였고(실시예 3-1 참조), 전이능 및 침습능 또한 크게 감소함을 구체적인 실험을 통해 확인하였다(실시예 3-2 참조).In one embodiment of the present invention, when the expression of Olig2 in melanoma cells was suppressed, it was confirmed that the cell viability was significantly reduced (see Example 3-1), and metastatic ability and invasive ability were also greatly reduced. was confirmed through a specific experiment (see Example 3-2).
본 발명의 다른 실시예에서는, Olig2의 발현을 억제한 흑색종 세포에 흑색종 치료제인 다브라페닙을 처리하는 경우, 다브라페닙의 효과를 증진시킴으로써, 흑색종의 생장을 억제할 수 있는 것을 확인하였다(실시예 4 참조).In another embodiment of the present invention, it was confirmed that melanoma growth can be inhibited by enhancing the effect of dabrafenib when treating melanoma cells in which the expression of Olig2 is suppressed, dabrafenib, a melanoma therapeutic agent. (see Example 4).
상기와 같은 결과를 통해 본 발명의 발명자들은 본 발명의 Olig2의 발현 또는 활성을 억제할 수 있는 억제제를 처리하는 경우, 흑색종의 전이 및 생장을 현저하게 억제할 수 있음을 구체적으로 확인했으며, 상기 억제제와 다브라페닙을 병용하여 투여하는 경우, 적은양으로도 다브라페닙의 효과인 흑색종 억제 효과를 증진시킬 수 있음을 확인하였다. 이와 같은 결과를 통해 Olig2 억제제는 흑색종 치료분야에서 폭넓게 활용될 수 있다는 사실을 확인할 수 있었다.Through the above results, the inventors of the present invention specifically confirmed that when an inhibitor capable of inhibiting the expression or activity of Olig2 of the present invention is treated, metastasis and growth of melanoma can be significantly inhibited, When the inhibitor and dabrafenib were administered in combination, it was confirmed that even a small amount could enhance the melanoma inhibitory effect of dabrafenib. Through these results, it was confirmed that the Olig2 inhibitor can be widely used in the field of melanoma treatment.
상기 약학적 조성물은 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용가능한 담체를 1종 이상 포함할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The pharmaceutical composition may include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
상기 약학적 조성물은 비경구, 피하, 정맥내, 근육내, 복강내, 경피내 또는 경구를 통해 병용하여 투여될 수 있다. 상기 약제는 경구 투여 또는 표적 기관에 직접적으로 투여될 수도 있으며, 투여량은 환자의 연령, 성별, 건강상태 및 체중, 병행치료의 종류, 투여시간, 투여방법, 배설률, 질환의 중증도, 치료의 빈도 및 원하는 효과의 성질 등에 비추어 결정될 수 있다. 또한, 치료되는 질환, 부위-특이적 치료의 필요성, 투여되는 약물의 양과 같은 조건들을 고려하여 전신적 또는 국소적으로 투여할 수 있다.The pharmaceutical composition may be administered in combination parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intradermally or orally. The drug may be administered orally or directly to a target organ, and the dosage may vary depending on the patient's age, sex, health condition and weight, type of concurrent treatment, administration time, administration method, excretion rate, disease severity, and frequency of treatment. and the nature of the desired effect. In addition, it may be administered systemically or locally in consideration of conditions such as the disease to be treated, the need for site-specific treatment, and the amount of the drug to be administered.
상기 약학적 조성물은 임의의 약학적 제형을 가질 수 있다. 예를 들면, 정제, 캡슐, 환제, 분말, 서방성 제형, 용액, 또는 현탁액으로서의 경구 투여에 적합한 형태이거나, 멸균 용액, 현탁액 또는 유화액으로서의 비경구 주사에 적합한 형태이거나, 연고 또는 크림으로서의 국소 투여에 적합한 형태이거나, 또는 좌제로서의 직장 투여에 적합한 형태일 수 있다. 상기 조성물은 정제, 환제, 주사제, 또는 이들의 조합일 수 있다. 상기 약학적 조성물은 정확한 투여량을 단일 투여하기에 적합한 단위 투여형일 수 있다.The pharmaceutical composition may have any pharmaceutical formulation. For example, in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulation, solution, or suspension, in a form suitable for parenteral injection as a sterile solution, suspension or emulsion, or in a form suitable for topical administration as an ointment or cream. It may be in a suitable form or may be in a form suitable for rectal administration as a suppository. The composition may be a tablet, pill, injection, or a combination thereof. The pharmaceutical composition may be in unit dosage form suitable for single administration of precise dosages.
상기 약학적 조성물을 제공하거나 약학적 조성물을 이용하여 질환의 치료를 수행함에 있어서, 상기 약학적 조성물은 단독으로 또는 조합하여, 또는 다른 치료제 또는 진단제와 조합하여 사용할 수 있다. 일 구체예에 있어서, 상기 약학적 조성물은 일반적으로 허용되는 의학적 관행에 따라 의도된 질환에 대해 전형적으로 처방되는 다른 약제와 동시 투여될 수 있다. 또한, 상기 약학적 조성물은 일반적으로 포유동물, 예를 들면, 인간의 생체내(in vivo)에서 이용될 수 있다.In providing the pharmaceutical composition or treating a disease using the pharmaceutical composition, the pharmaceutical composition may be used alone or in combination, or in combination with other therapeutic agents or diagnostic agents. In one embodiment, the pharmaceutical composition may be administered concurrently with other agents typically prescribed for the intended disease in accordance with generally accepted medical practice. In addition, the pharmaceutical composition can generally be used in a mammal, for example, a human in vivo (in vivo).
또한, 본 발명은 BRAF 억제제 및 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 또는 활성 억제제를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 흑색종의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating melanoma, comprising administering a BRAF inhibitor and an Olig2 (Oligodendrocyte transcription factor 2) gene or protein expression or activity inhibitor to an individual in need thereof.
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다.As used herein, the term “administration” means providing a given composition of the present invention to a subject by any suitable method.
본 발명에서 사용되는 용어 "개체"란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는, 인간 또는 비-인간인 영장류, 생쥐(mouse), 개, 고양이, 말, 및 소 등의 포유류를 의미한다.As used herein, the term "subject" refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, dogs, cats, horses, and cattle. means mammals.
또 다른 양상은 흑색종의 예방 또는 치료용 약제의 제조를 위한 BRAF 억제제 및 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 또는 활성 억제제의 용도를 제공한다.Another aspect provides the use of a BRAF inhibitor and an Oligodendrocyte transcription factor 2 (Olig2) gene or inhibitor of expression or activity for the manufacture of a medicament for the prophylaxis or treatment of melanoma.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1. 실험준비 및 실험방법Example 1. Experimental preparation and experimental method
1-1. 세포배양1-1. cell culture
인간 흑색종 세포인 A735, 501mel 및 HM3KO 세포를 10% FBS(Fetal Bovine Serum)(Gibco, Los Angeles, CA, USA) 및 1% 페니실린/스트렙토마이신(penicillin/streptomycin)이 보충된 Dulbecco`s modified eagle`s medium(DMEM; Welgene Inc., 한국)에서 배양한다. 인간 흑색종 세포인 MNT1는 20% FBS, 1% 페니실린/스트렙토마이신 및 20mM 하이드록시에틸 피페라진에탄설폰산(hydroxyethyl piperazineethanesulfonic, HEPES)이 보충된 최소 필수 배지(minimum essential media, MEM; Welgene Inc., Korea)에서 배양한다. 정상 인간 멜라닌 세포인 NHEM는 인간 멜라닌 세포 성장 보충제(Human Melanocyte Growth Supplement, HMGS; Invitrogen Life Technologies, Carlsbad, CA, USA) 및 1%의 페니실린/스트렙토마이신을 함유하는 배지 254(Medium 254, M-254-500; Invitrogen Life Technologies, Carlsbad, CA, USA)에서 37℃ 및 5%의 CO2의 습한 조건(humidified atmosphere)에서 배양한다.Human melanoma cells, A735, 501mel and HM3KO cells, were treated with Dulbecco's modified eagle supplemented with 10% Fetal Bovine Serum (Gibco, Los Angeles, CA, USA) and 1% penicillin/streptomycin. Culture in `s medium (DMEM; Welgene Inc., Korea). MNT1, a human melanoma cell, was prepared in minimum essential media (MEM; Welgene Inc.) supplemented with 20% FBS, 1% penicillin/streptomycin and 20 mM hydroxyethyl piperazineethanesulfonic acid (HEPES). Korea). Normal human melanocytes, NHEMs, were prepared from Human Melanocyte Growth Supplement (HMGS; Invitrogen Life Technologies, Carlsbad, CA, USA) and medium 254 (Medium 254, M-254) containing 1% penicillin/streptomycin. -500; Invitrogen Life Technologies, Carlsbad, CA, USA) in a humidified atmosphere of 37° C. and 5% CO 2 .
1-2. 세포 생존율 확인1-2. Check cell viability
세포 생존율을 확인하기 위하여, 세포를 96 웰-플레이트에 1x104개 세포/웰로 시딩(seeding)한 다음, 각각의 세포에 CRISPR-Olig2를 형질감염 시켰고, 24시간, 48시간 및 72시간이 경과한 다음, 세포 생존율을 확인하였다. 세포 생존율을 확인하기 위하여 Ez-Cytox assay kit(Daeil Lab Service Co Ltd, Seoul, Korea)을 활용하였고, 구체적으로 10μl의 Ez-Cytox 용액을 각 웰-플레이트에 첨가한 다음, 웰-플레이트를 5% CO2 조건 및 37℃의 인큐베이터에서 추가로 1시간 동안 인큐베이션했다. 세포 생존력은 ELISA 판독기(Tecan, Mannedorf, Switzerland)에 의해 490nm 파장에서 확인하였고, 상대적 세포독성은 세포의 생존력을 백분율로 표현하여 측정한다.To check cell viability, cells were seeded at 1x10 4 cells/well in a 96-well-plate, and then, each cell was transfected with CRISPR-Olig2, and 24 hours, 48 hours and 72 hours had elapsed. Next, cell viability was checked. To check the cell viability, the Ez-Cytox assay kit (Daeil Lab Service Co Ltd, Seoul, Korea) was used. Specifically, 10 μl of Ez-Cytox solution was added to each well-plate, and then the well-plate was added to 5% Incubated for an additional 1 hour under CO 2 conditions and in an incubator at 37°C. Cell viability was confirmed at 490 nm wavelength by an ELISA reader (Tecan, Mannedorf, Switzerland), and relative cytotoxicity was measured as a percentage of cell viability.
1-3. 세포 계수 분석1-3. Cell count analysis
세포를 96-웰 플레이트에 1x104 세포/웰로 시딩한 다음, CRISPR/Cas9 플라스미드를 활용하여 형질감염을 시키고 72시간이 경과한 다음, 세포에 트립신(trypsin)을 처리했다. 트리판 블루 염료(Sigma Aldrich, St. Louis, MO, USA) 10ul를 세포 현탁액과 동일하게 혼합하였다. 트리판블루염료(trypan blue dye)로 세포를 염색한 다음, 혈구계산기(hemocytometer)를 이용하여 각 샘플에 3회 반복하여 세포수를 세었다.Cells were seeded in a 96-well plate at 1× 10 4 cells/well, and then transfected using CRISPR/Cas9 plasmid. After 72 hours, the cells were trypsin-treated. 10 ul of trypan blue dye (Sigma Aldrich, St. Louis, MO, USA) was mixed in the same manner as the cell suspension. After staining the cells with trypan blue dye, each sample was repeated three times and the number of cells was counted using a hemocytometer.
1-4. 실시간 중합효소연쇄반응(Real time polymerase chain reaction) 분석1-4. Real time polymerase chain reaction analysis
RT-PCR 분석을 위하여 세포를 6 웰 플레이트에 5x105 세포/웰로 시딩한다. 전체 RNA(Total RNA)를 추출하기 위해 실시예 1-1에서 준비한 흑색종 세포에 TRIZOL 시약(Takara Bio, Inc., Tokyo, Japan)을 처리한 다음, TRIZOL의 1/5 수준의 클로로포름(Chloroform, CHCl3, 99%, Junsei Chemical, Japan)을 처리하여 혼합한다. Vortex mixer를 혼합한 후, 4℃의 온도에서 13,000rpm로 15 분 동안 원심분리한다. 투명한 상층액의 일정량을 취하여 동량의 이소프로판올(isopropanol)과 혼합하였다. 튜브 원심분리기를 4℃, 13,000rpm, 20분에서 인버팅(inverting)시킨 후. 상층액을 제거하고 남은 펠렛을 디에틸 파이로카보네이트 처리수(Diethyl pyrocarbonate treated water)로 희석했다. For RT-PCR analysis, cells are seeded at 5x10 5 cells/well in 6 well plates. To extract total RNA (Total RNA), the melanoma cells prepared in Example 1-1 were treated with TRIZOL reagent (Takara Bio, Inc., Tokyo, Japan), and then 1/5 of TRIZOL in chloroform (Chloroform, CHCl 3 , 99%, Junsei Chemical, Japan) was treated and mixed. After mixing the Vortex mixer, centrifuged for 15 minutes at 13,000 rpm at a temperature of 4 ℃. A certain amount of the transparent supernatant was taken and mixed with the same amount of isopropanol. After inverting the tube centrifuge at 4° C., 13,000 rpm, 20 min. The supernatant was removed and the remaining pellet was diluted with diethyl pyrocarbonate treated water.
열 순환기 SimpliAmp™, Thermo Fisher Scientific, MA, USA)를 사용하여 특정 프라이머를 사용하여 역전사 중합효소 연쇄 반응(RT-PCR)에 의해 상보적 DNA(cDNA)의 합성을 수행했고, 이를 위해 사용된 프라이머의 구체적인 서열은 하기 표 1에 나타내었다. 합성된 cDNA는 1X Tris Acetate-EDTA(TAE) 완충액(Elpis Biotech, 대전, 한국) 및 RedSafe™ Nucleic에서 2% agarose gel(Bioneer Co., 대전, 한국)에서 Acid Staining Solution(iNtRON Biotechnology, 한국 성남)를 사용하여 시각화 하였다.Synthesis of complementary DNA (cDNA) was performed by reverse transcription polymerase chain reaction (RT-PCR) using specific primers using a thermocycler SimpliAmp™, Thermo Fisher Scientific, MA, USA), and the primers used for this purpose The specific sequence of is shown in Table 1 below. The synthesized cDNA was prepared in 1X Tris Acetate-EDTA (TAE) buffer (Elpis Biotech, Daejeon, Korea) and 2% agarose gel (Bioneer Co., Daejeon, Korea) in RedSafe™ Nucleic Acid Staining Solution (iNtRON Biotechnology, Seongnam, Korea) was visualized using
| GeneGene |
Sequence of primerssequence of primers
(5'→ 3`)(5' → 3') |
TmTm
(℃)(℃) |
AmpAmp
cyclescycles |
ProductProduct
sizesize |
|
Olig2Olig2
(human)(human) |
F: GAATCCGCTGGTATCCACGAF: GAATCCGCTGGTATCCACGA | 5555 | 3333 | 335bp335bp |
| R: GCGGAGCGAGACGTTTAGAAR: GCGGAGCGAGACGTTTAGAA | ||||
|
β-actinβ-actin
(human)(human) |
F: GGCATCGTGATGGACTCCGF: GGCATCGTGATGGACTCCG | 5959 | 2727 | 610bp610bp |
| R: GCTGGAAGGTGGACAGCGAR: GCTGGAAGGTGGACAGCGA |
1-5. 상처 치유 분석(wound healing assay)1-5. wound healing assay
흑색종 세포를 12 웰 플레이트에 1 x 105개 세포/웰 수준으로 시딩한 다음, CRISPR/Cas9 플라스미드를 세포에 형질감염 시키고, 세포가 플레이트의 약 90% 정도를 차지하게 되었을 때, 노란색 마이크로피펫 팁으로 각 웰의 중앙을 가로지르면서 단층을 부드럽게 긁어 상처를 내고, 인산완충식염수(PBS)로 두 번 세척한다. 표시된 시간에 상처의 치유가 확인되었으며, 이에 대한 이미지는 현미경으로 촬영하였다. 모든 실험은 세 번 반복되었다. 상처 간극 면적의 차이는 ImageJ 소프트웨어 1.45s(U.S. National Institutes of Health, Bethesda, MD, USA)를 통해 대조군과 비교하여 정량화하였다. 면적 측정은 0시간의 면적 값에서 24시간과 48시간의 면적 값을 뺀 값을 대조군의 백분율로 환산하여 계산하였다.Melanoma cells were seeded in 12-well plates at 1 x 10 5 cells/well, then CRISPR/Cas9 plasmids were transfected into the cells, and when the cells occupied approximately 90% of the plate, a yellow micropipette Wound the monolayer by gently scraping it across the center of each well with a tip, and wash twice with phosphate-buffered saline (PBS). Healing of the wound was confirmed at the indicated time, and the image was taken with a microscope. All experiments were repeated three times. The difference in wound gap area was quantified compared to the control group through ImageJ software 1.45s (US National Institutes of Health, Bethesda, MD, USA). The area measurement was calculated by converting the area value at 0 hour by subtracting the area value at 24 hours and 48 hours into a percentage of the control group.
1-6. 트랜스웰(transwell) 침습성 및 이동성 분석1-6. Transwell invasiveness and mobility assays
시험관내 침입 분석을 위해, 흑색종 세포들을 12 웰 플레이트에 접종한 다음, CRISPR/Cas9 플라스미드의 형질감염 24시간 후에 세포를 트립신 처리하였다. 세포를 1% FBS를 함유하는 DMEM 배지에서 8.0μm 기공 크기의 폴리카보네이트 막(polycarbonate membranes; Corning Incorporated, Corning, NY, USA)으로 각 챔버당 5 x 104개의 세포로 다시 시딩한 다음, 18시간 동안 인큐베이션하였다. 상기 챔버는 구체적으로 상부 챔버는 무혈청 DMEM 배지로 희석된 Matrigel®(Corning, Kaiserslautern, Germany)로 코팅하였으며, 1% 젤라틴으로 상부 챔버의 바닥을 코팅했다. 바닥 챔버는 10% FBS를 포함하는 DMEM 배지로 채웠다. Matrigel이 코팅된 막을 통해 침입한 세포를 10% 포르말린에 고정하고 PBS로 2회 세척하였다. 침입한 세포의 핵을 시각화하기 위하여 위해 4',6-diamidino-2-phenylindole(DAPI)로 염색했다. 침습성 세포는 x100 배율에서 형광 현미경에 의해 검출되었다. ImageJ 소프트웨어 1.45s(U.S. National Institutes of Health, Bethesda, MD, USA)를 사용하여 염색된 핵을 계수하고 정량화하였다.For in vitro invasion assays, melanoma cells were seeded in 12 well plates and then cells were trypsinized 24 h after transfection with CRISPR/Cas9 plasmids. Cells were re-seeded with polycarbonate membranes (Corning Incorporated, Corning, NY, USA) with 8.0 μm pore size in DMEM medium containing 1% FBS at 5 x 10 4 cells per chamber, followed by 18 h. incubated for a while. Specifically, the upper chamber was coated with Matrigel® (Corning, Kaiserslautern, Germany) diluted with serum-free DMEM medium, and the bottom of the upper chamber was coated with 1% gelatin. The bottom chamber was filled with DMEM medium containing 10% FBS. Cells that invaded through the Matrigel-coated membrane were fixed in 10% formalin and washed twice with PBS. In order to visualize the nucleus of the invaded cells, it was stained with 4',6-diamidino-2-phenylindole (DAPI). Invasive cells were detected by fluorescence microscopy at x100 magnification. Stained nuclei were counted and quantified using ImageJ software 1.45s (US National Institutes of Health, Bethesda, MD, USA).
실시예 2. CRISPR-Cas9 기술을 활용한 Oligodendrocyte transcription factor 2 발현 억제 세포 모델의 제조Example 2. Preparation of Oligodendrocyte transcription factor 2 expression suppression cell model using CRISPR-Cas9 technology
Olig2 억제제의 효과를 확인하기 위해, 악성 흑색종 세포인 501mel 세포에 CRISPR-Cas9 기법을 활용하여 전이인자 Olig 2(Oligodendrocyte transcription factor 2)의 발현이 억제된 세포를 제작하였다. 구체적으로 상기 실시예 1-1에서 준비한 세포에 UltraCruz 형질감염 시약(UltraCruz Transfection Reagent(Santacruz Biotechnology, Dallas, TX, USA))을 사용하여 12시간 동안 20nt guide sgRNA 및 Cas9 뉴클레아제를 인코딩하는 3개의 상이한 gRNA 플라스미드로 구성되어 있는 sc-400670-KO-2: Olig2 CRISPR/Cas9 KO 플라스미드(h2)(Santacruz Biotechnology, Dallas, TX, USA)로 형질감염시켰다. 상기 플라스미드의 3가지 gRNA는 gRNA1: 5`-GGCTGCGTCGTCCACCAAGA-3`, gRNA2: 5`-CACCTCGTCGTCTACGTCGT-3` 또는 gRNA3: 5`-CGCCCATAGCCGACACGTGG-3` 일 수 있다.To confirm the effect of the Olig2 inhibitor, CRISPR-Cas9 technique was used in 501mel cells, a malignant melanoma cell, to prepare cells in which the expression of the transfer factor Olig 2 (Oligodendrocyte transcription factor 2) was suppressed. Specifically, using UltraCruz transfection reagent (UltraCruz Transfection Reagent (Santacruz Biotechnology, Dallas, TX, USA)) for the cells prepared in Example 1-1, 20nt guide sgRNA and Cas9 nuclease encoding three The sc-400670-KO-2 consisting of different gRNA plasmids: Olig2 CRISPR/Cas9 KO plasmids (h2) (Santacruz Biotechnology, Dallas, TX, USA) were transfected. The three gRNAs of the plasmid may be gRNA1: 5'-GGCTGCGTCGTCCACCAAGA-3', gRNA2: 5'-CACCTCGTCGTCTACGTCGT-3' or gRNA3: 5'-CGCCCATAGCCGACACGTGG-3'.
상기 방법으로 제작한 501 mel 세포의 Olig 2 발현 수준을 확인한 결과, 도 1에 나타낸 바와 같이, Olig2 gRNA의 처리 농도에 비례하여 Olig2의 단백질 발현 수준이 억제되는 Olig2 억제 세포 모델을 제작되었음을 확인하였다.As a result of confirming the Olig 2 expression level of the 501 mel cells prepared by the above method, as shown in FIG. 1 , it was confirmed that an Olig2 suppressed cell model in which the protein expression level of Olig2 was suppressed in proportion to the treatment concentration of Olig2 gRNA was prepared.
실시예 3. Oligodendrocyte transcription factor 2 발현이 억제된 흑색종 세포의 변화 확인Example 3. Confirmation of changes in the expression of oligodendrocyte transcription factor 2 suppressed melanoma cells
3-1. 생존율 확인 3-1. Check the survival rate
암세포는 정상세포와 달리 무한히 증식하는 특성을 가지고 있으므로, Olig2의 발현이 억제되어 있는 악성 흑색종 세포의 증식율을 측정하였다. 보다 구체적으로, 상기 실시예 2에서 제조한 Olig2의 발현이 억제되어 있는 악성 흑색종 세포(A375 세포 및 501mel 세포)에서 Olig2 gRNA의 처리 농도 및 시간에 따른 Ez-CyToX 세포 생존력 분석하였다. Since cancer cells have a characteristic of infinitely proliferating unlike normal cells, the proliferation rate of malignant melanoma cells in which Olig2 expression is suppressed was measured. More specifically, in the malignant melanoma cells (A375 cells and 501mel cells) in which the expression of Olig2 prepared in Example 2 is suppressed, Ez-CyToX cell viability was analyzed according to the concentration and time of treatment with Olig2 gRNA.
단백질이 억제된 악성 흑색종 세포 모델의 1일, 2일, 3일차 세포 생존율을 확인한 결과, 도 2a(A375 세포), 도 2b(501mel 세포) 및 하기 표 2(501 mel 세포 데이터)에 나타낸 바와 같이 세포 생존율이 유의한 수준으로 감소하는 것을 확인하였다. 세포계수 분석을 통해서 세포수를 확인했을 때에도, 도 2c(A375 세포) 및 도 2d(501mel 세포)에 나타낸 바와 같이, 유의한 수준으로 세포수가 감소한 것을 확인하였다. As a result of confirming the cell viability on the 1st, 2nd, and 3rd day of the protein-suppressed malignant melanoma cell model, as shown in Figure 2a (A375 cells), Figure 2b (501mel cells) and Table 2 (501 mel cell data) Likewise, it was confirmed that the cell viability decreased to a significant level. Even when the number of cells was confirmed through cytometry analysis, it was confirmed that the number of cells decreased to a significant level, as shown in FIGS. 2c (A375 cells) and 2d (501mel cells).
|
Con |
CRISPR-Olig2 1μg1 μg of CRISPR-Olig2 | CRISPR-Olig2 2μgCRISPR-Olig2 2μg | ||
| % of Con% of Con | Day1Day1 | 100.00100.00 | 87.4587.45 | 86.6386.63 |
| Day2Day2 | 100.00100.00 | 86.1486.14 | 73.9073.90 | |
| Day3Day3 | 100.00100.00 | 76.6676.66 | 71.0271.02 |
3-2. 전이성 확인3-2. metastatic confirmation
악성 흑색종의 주요 특징 중 하나인 전이는 원발성 종양에서 세포가 분리되어 이리저리 움직이는 현상으로, 이동성이 있는 흑생종 세포는 혈관과 림프관을 순환하여 다른 말단 기관에 정착하여 이차 종양을 형성하고 결국엔 전이된다(NGUYEN, D.X., BOS, P.D. and MASSAGUE, J., 2009. Metastasis: from dissemination to organ-specific colonization. Nature Reviews Cancer,
9(4), pp. 274.)Metastasis, one of the main characteristics of malignant melanoma, is a phenomenon in which cells are separated from the primary tumor and move around. Migrating melanoma cells circulate in blood and lymph vessels, settle in other end organs, form secondary tumors, and eventually metastasize. (NGUYEN, DX, BOS, PD and MASSAGUE, J., 2009. Metastasis: from dissemination to organ-specific colonization. Nature Reviews Cancer, 9 (4), pp. 274.)
Olig2가 억제된 세포(A375, 501mel)에서 흑색종의 이동성(전이성) 및 침습성의 변화가 일어나는지 여부를 확인하기 위하여 상기 실시예 1-5(상처 치유 분석)을 사용하여 세포 이동을 평가하고, 실시예 1-6(트랜스웰 분석)을 수행하였다. Cell migration was evaluated using Examples 1-5 (wound healing assay) above to determine whether changes in the migration (metastatic) and invasiveness of melanoma occurred in Olig2-inhibited cells (A375, 501mel) Examples 1-6 (transwell analysis) were performed.
그 결과, 도 3a(A375세포) 및 도 4a(501 mel세포)에 나타낸 바와 같이, 대조군 대비 24시간 및 48시간 경과에 따라 세포가 상처부위로 이동이 억제되는 것을 확인하였다. 보다 구체적으로, 상처 간극의 면적 차이를 대조군과 비교하여 정량화한 결과, 도 3b에 나타낸 바와 같이, A375 세포의 경우, 24시간이 경과했을 때, Olig2 억제를 위한 gRNA 1㎍를 처리한 그룹에서 9 ± 10.0% 감소, gRNA 2㎍을 처리한 그룹에서 16 ± 7.5% 감소했고, 48시간이 경과했을 때, gRNA 1㎍를 처리한 그룹에서 51 ± 3.8% 감소, gRNA 2㎍를 처리한 그룹에서 62 ± 16.5% 감소함을 확인할 수 있었다. 또한, 도 4b에 나타낸 바와 같이, 501mel 세포의 경우, 24시간이 경과했을 때, Olig2 억제를 위한 gRNA 1㎍를 처리한 그룹에서 8 ± 9.5% 감소, gRNA 2㎍을 처리한 그룹에서 18 ± 17.0% 감소했고, 48시간이 경과했을 때, gRNA 1㎍를 처리한 그룹에서 28 ± 18.6% 감소, gRNA 2㎍를 처리한 그룹에서 55 ± 10.3%% 감소함을 확인할 수 있었다.As a result, as shown in FIGS. 3a (A375 cells) and 4a (501 mel cells), it was confirmed that the cells were inhibited from moving to the wounded area according to the lapse of 24 hours and 48 hours compared to the control group. More specifically, as a result of quantifying the difference in the area of the wound gap compared to the control group, as shown in FIG. 3b , in the case of A375 cells, when 24 hours elapsed, in the group treated with 1 μg of gRNA for Olig2 inhibition, 9 ± 10.0% reduction, 16 ± 7.5% reduction in the group treated with 2 μg gRNA, and 51 ± 3.8% reduction in the group treated with gRNA 1 μg at 48 hours, 62 in the group treated with 2 μg gRNA It was confirmed that the decrease was ± 16.5%. In addition, as shown in Figure 4b, in the case of 501mel cells, when 24 hours elapsed, a decrease of 8 ± 9.5% in the group treated with 1 μg of gRNA for Olig2 inhibition, 18 ± 17.0 in the group treated with 2 μg of gRNA % decreased, and when 48 hours elapsed, it was confirmed that the group treated with gRNA 1㎍ was reduced by 28 ± 18.6%, and in the group treated with gRNA 2㎍, it was reduced by 55 ± 10.3%%.
다음으로, 트랜스웰 전이 분석을 수행하여 세포의 이동성을 분석하였다. 세포를 상부 챔버에 접종하고 18시간 동안 트랜스웰 챔버를 통해 이동하도록 하였다. 그 후, 챔버의 기공을 통해 이동한 세포를 DAPI로 염색하고 현미경으로 관찰(도 5a 참조)하고, Image J 프로그램을 사용하여 염색된 핵을 계수하고 정량화한 결과, 도 5b에 나타낸 바와 같이, 18시간이 경과했을 때, Olig2 억제를 위한 gRNA를 1μg 처리한 그룹에서 63 ± 5.2% 감소, gRNA를 2μg 처리한 그룹에서 58 ± 5.0% 감소함을 확인하였다. Next, transwell metastasis assay was performed to analyze cell mobility. Cells were seeded into the upper chamber and allowed to migrate through the transwell chamber for 18 hours. Thereafter, the cells that migrated through the pores of the chamber were stained with DAPI and observed under a microscope (see Fig. 5a), and the stained nuclei were counted and quantified using the Image J program. As shown in Fig. 5b, 18 As time elapsed, it was confirmed that a 63 ± 5.2% decrease in the group treated with 1 μg of gRNA for Olig2 inhibition and 58 ± 5.0% decreased in the group treated with 2 μg of gRNA.
흑색종 세포의 침습(invasion)능력을 확인하기 위하여, 트랜스웰 막 챔버의 세포외 기질 대신에 matrigel을 0% FBS DMEM 배지로 희석한 다음, 상부챔버에 코팅하였고, matrigel을 통해 침투한 세포를 DAPI로 염색하여 현미경으로 관찰(도 6a 참조)하고, Image J 프로그램을 사용하여 염색된 핵을 계수하고 정량화한 결과, 도 6b에 나타낸 바와 같이, 상대적인 세포의 침습은 A375세포에서 Olig2 억제를 위한 gRNA를 1㎍처리한 그룹에서 48 ± 8.2% 감소, gRNA 2㎍ 처리한 그룹에서 54 ± 8.6%까지 감소함을 확인할 수 있었다.To confirm the invasion ability of melanoma cells, instead of the extracellular matrix of the transwell membrane chamber, matrigel was diluted with 0% FBS DMEM medium, and then coated on the upper chamber. was stained with and observed under a microscope (see Fig. 6a), and the stained nuclei were counted and quantified using the Image J program. It was confirmed that the decrease was 48 ± 8.2% in the group treated with 1 μg, and decreased by 54 ± 8.6% in the group treated with gRNA 2 μg.
본 발명자들은 상기와 같은 결과를 통해 Olig2를 억제하는 경우, 흑색종의 전이를 유의한 수준으로 억제할 수 있음을 확인하였다.The present inventors confirmed that when Olig2 is inhibited through the above results, metastasis of melanoma can be inhibited to a significant level.
실시예 4. Oligodendrocyte transcription factor 2 발현 억제된 세포에 Dabrafenib 처리시 악성 흑색종 세포의 생존율 확인Example 4. Confirmation of survival rate of malignant melanoma cells when oligodendrocyte transcription factor 2 expression is suppressed by Dabrafenib treatment
본 발명의 Olig2 조성물의 병용투여 효과를 구체적으로 확인하기 위해, 기존에 이미 B-Raf inhibitor로서 미 FDA에서 승인된 흑색종 치료제인 다브라페닙(Dabrafenib)을 정상세포 또는 Olig2 발현이 억제되어 있는 501mel 세포에 처리하고 3일이 경과한 다음, 세포 생존율을 확인하였다. 그 결과, 도 7 및 하기 표 3에 나타낸 바와 같이, Olig2의 발현이 억제되어 있는 악성 흑색종 세포에 다브라페닙을 투여한 경우, Olig2가 발현되고 있는 흑색종 세포에 다브라페닙을 처리한 경우보다 흑색종 세포의 생장 억제효과가 뛰어남을 확인하였다.In order to specifically confirm the effect of co-administration of the Olig2 composition of the present invention, Dabrafenib, a melanoma treatment previously approved by the US FDA as a B-Raf inhibitor, was used in normal cells or 501mel in which Olig2 expression is suppressed. After 3 days of treatment with the cells, the cell viability was checked. As a result, as shown in FIG. 7 and Table 3 below, when dabrafenib was administered to malignant melanoma cells in which Olig2 expression was suppressed, and when dabrafenib was treated to Olig2-expressing melanoma cells It was confirmed that the growth inhibitory effect of melanoma cells was superior.
| ConCon | CRISPR-Olig2CRISPR-Olig2 | DabrafenibDabrafenib |
CRISPR-Olig2CRISPR-Olig2
+ Dabrafenib+ Dabrafenib |
|
| % of Con% of Con | 100.00100.00 | 72.2972.29 | 60.8960.89 | 49.3449.34 |
상기와 같은 결과를 통해, 본 발명의 Olig2 억제제를 기존에 흑색종 치료제로 활용되고 있는 다브라페닙과 병용하여 투여하는 경우 다브라페닙 단독으로 투여하는 경우보다 흑색종의 생장을 보다 효과적으로 억제할 수 있다는 사실을 확인할 수 있었다. Through the above results, when the Olig2 inhibitor of the present invention is administered in combination with dabrafenib, which has been previously used as a treatment for melanoma, it is possible to more effectively inhibit the growth of melanoma than when dabrafenib is administered alone. I could confirm that there was.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
Claims (9)
- BRAF 억제제 및 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 또는 활성 억제제를 포함하는, 흑색종의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating melanoma, comprising an inhibitor of BRAF inhibitor and Olig2 (Oligendrocyte transcription factor 2) gene or protein expression or activity inhibitor.
- 제1항에 있어서,According to claim 1,상기 BRAF 억제제는 다브라페닙(dabrafenib)인 것을 특징으로 하는, 약학적 조성물.The BRAF inhibitor is dabrafenib (dabrafenib), characterized in that the pharmaceutical composition.
- BRAF 억제제 및 Olig2(Oligodendrocyte transcription factor 2) 유전자 또는 단백질의 발현 또는 활성 억제제를 포함하는, 흑색종의 전이 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating metastasis of melanoma, comprising an inhibitor of BRAF inhibitor and Olig2 (Oligendrocyte transcription factor 2) gene or protein expression or activity.
- BRAF 억제제와 병용되는 흑색종 예방 또는 치료 효과 증진용 약학적 조성물로서, 상기 조성물은 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 또는 활성 억제제를 유효성분으로 포함하는 것을 특징으로 하는, 약학적 조성물.A pharmaceutical composition for enhancing the effect of preventing or treating melanoma used in combination with a BRAF inhibitor, wherein the composition comprises an inhibitor of expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient, A pharmaceutical composition.
- 제4항에 있어서,5. The method of claim 4,상기 BRAF 억제제는 다브라페닙(dabrafenib)인 것을 특징으로 하는, 약학적 조성물.The BRAF inhibitor is dabrafenib (dabrafenib), characterized in that the pharmaceutical composition.
- 제4항에 있어서, 5. The method of claim 4,상기 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 억제제는 전사인자 Olig2(Oligodendrocyte transcription factor 2) mRNA에 특이적인 안티센스 올리고뉴클레오티드, 작은 간섭 RNA(small interfering RNA; siRNA), 짧은 헤어핀 RNA(short hairpin RNA; shRNA) 및 리보자임(ribozyme)으로 구성된 군으로부터 선택되는 어느 하나인 것을 특징으로 하는, 약학적 조성물.The transcription factor Olig2 (Oligodendrocyte transcription factor 2) expression inhibitors include an antisense oligonucleotide specific for the transcription factor Olig2 (Oligodendrocyte transcription factor 2) mRNA, small interfering RNA (siRNA), short hairpin RNA; shRNA) and ribozyme (ribozyme), characterized in that any one selected from the group consisting of, a pharmaceutical composition.
- 제4항에 있어서,5. The method of claim 4,상기 전사인자 Olig2(Oligodendrocyte transcription factor 2) 단백질의 활성 억제제는 전사인자 Olig2(Oligodendrocyte transcription factor 2) 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 미메틱스, 앱타머, 항체 및 천연물로 구성된 군으로부터 선택되는 어느 하나인 것인, 약학적 조성물.The transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein activity inhibitor is from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and natural products that specifically bind to transcription factor Olig2 (Oligodendrocyte transcription factor 2) protein. Any one selected, the pharmaceutical composition.
- 제4항에 있어서,5. The method of claim 4,상기 병용은 동시에(simultaneous), 별도로(separate) 또는 순차적(sequential) 투여되는 것을 특징으로 하는, 조성물.The composition is characterized in that the combination is administered simultaneously (simultaneous), separately (separate) or sequentially (sequential).
- BRAF 억제제와 병용되는 흑색종의 전이 예방 또는 치료 효과 증진용 약학적 조성물로서, 상기 조성물은 전사인자 Olig2(Oligodendrocyte transcription factor 2)의 발현 또는 활성 억제제를 유효성분으로 포함하는 것을 특징으로 하는, 약학적 조성물.A pharmaceutical composition for preventing metastasis or enhancing the therapeutic effect of melanoma used in combination with a BRAF inhibitor, wherein the composition comprises an inhibitor of the expression or activity of transcription factor Olig2 (Oligendrocyte transcription factor 2) as an active ingredient, a pharmaceutical composition.
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| KR1020210124582A KR102710867B1 (en) | 2020-12-18 | 2021-09-17 | A pharmaceutical composition for enhancing the therapeutic effect of melanoma comprising an oligodendrocyte transcription factor 2 inhibitor as an active ingredient |
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